PROCESSING AND PRODUCTS

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1 PROCESSING AND PRODUCTS The effect of dry-cured salt contents on accumulation of non-volatile compounds during dry-cured goose processing C. Y. Zhou,,2 Y. Wang,,2 J. X. Cao,,1 Y. J. Chen, Y. Liu, Y. Y. Sun, D. D. Pan, and C. R. Ou Key Laboratory of Animal Protein Food Processing Technology of Zhejiang Province, Ningbo University, Ningbo , China; Department of Food Science, Nanjing University of Finance & Economics, Nanjing , China; and College of Food Science and Technology, Shanghai Ocean University, Shanghai, , China ABSTRACT Twenty-four Eastern Zhejiang White Geese were slaughtered, dry-cured by 2 different kinds of salt contents (12 geese with 4% low salt level [LS]; 12 geese with 8% high salt level [HS]) for one d, marinated in brine for one d, and air dry-ripened at 16 C for 7 d. The effect of dry-curing salt contents on the changes in myofibril proteins, potential proteolysis activities, and total free amino acid (TFAA) in dry-cured goose was investigated. Compared to the raw, cathepsin B+L and calpains activities decreased at the end of dry-curing and the third d of dry-ripening. At the final products, the activities of cathepsin B+L and calpains were about half of those in raw meat. There was no difference in proteolysis activities except for the end of dry-curing (P < 0.05) and the 3 d of dry-ripening (P < 0.05) for cathepsin B+L, and the end of dry-curing (P < 0.05) for calpains (P < 0.05) between groups. Myosin light chain (MLC) and troponin-i were cleaved. Compared to the raw, TFAA increased by and 31.82% in the final products for HS (P < 0.001) and LS (P < 0.01), respectively. The increase of TFAA could be attributed to the proteolysis of myofibril proteins and retained proteolysis activities. No significant difference on TFAA and MLC and troponin-i bands was observed between groups in final products. This means that different proteolysis activities during processing did not cause the difference in quality of final products between groups, and that 4% low salt can be used in future applications. Key words: cathepsin B+L, calpains, free amino acids, myofibril proteins, dry-cured goose meat 2016 Poultry Science 95: INTRODUCTION Dry-cured goose, one of China s traditional meat products, which has a special shape, rose color, and delicate flavor, was produced from fresh goose meat by dry-curing, marinating, and air dry-ripening. The drycured goose is very popular in most Chinese provinces. The flavor, one of the most characteristic of dry-cured meat products, is made up of volatile and non-volatile compounds (Soldo et al., 2003). The mechanism of accumulation on volatile compounds and its flavor composition has been studied intensively. To our knowledge, the mechanism is from fatty acid and carbonyl compounds, such as ketones, aldehydes, esters, and alcohols, by hydrolyzing the lipid during processing (Sforza et al., 2006). The main volatile compounds in drycured meat products have been suggested to be Heptan- 1-alcohol, hexanal, 1- octene- 3- alcohol, methyl ke- C 2016 Poultry Science Association Inc. Received September 25, Accepted March 4, Corresponding author: caojinxuan@nbu.edu.cn 2 Both authors contributed equally to this work. tone, 1, 3-pentene- 3- alcohol, fatty acid, ethyl ester, et al. (Lorenzo et al., 2012; Wu et al., 2015), while free amino acids hydrolyzed from proteins by endogenous enzymes and exogenous enzymes are the primary nonvolatile composition. The accumulation and mechanism of volatile compounds in Chinese traditional dry-cured goose have been reported in our previous study (Ying et al., 2016). Up to now, the production of non-volatile components has not been evaluated. Free amino acids hydrolyzed from cytoskeletal proteins have been demonstrated to be the main nonvolatile components and the important precursors of volatile components (Hidalgo and Zamora, 2004). Calpains and cathepsin B & L were believed to play a major role in hydrolyzing myofibril proteins and have been extensively investigated by meat scientists (Ladrat et al., 2003). Cathepsin B+L have been demonstrated to retain high hydrolyzed activities for their substrates during the whole processing of dry-cured Jinhua hams and Teruel hams (Zhao et al., 2005; Larrea et al., 2006). Sárraga et al. (1993) reported that calpains lost most of their activities after dry-ripening for several months in 2160

2 THE FORMATION OF TASTED COMPOUNDS OF DRY-CURED GOOSE 2161 Iberian ham. The relative contribution of calpains was restricted to the first wk of processing due to their low stability even though they could contribute to an initial breakdown of the Z-disc (Rosell and Toldrá, 1996). Compared to dry-cured hams, the processing phase of Chinese traditional dry-cured goose can be completed within 2 wk. The specific role and activities of calpains and cathepsin B & L during processing in Chinese traditional dry-cured goose are still unclear. In recent years, dry-cured goose was still processed in a traditional way with high curing salt content (about 8%) in most of the rural areas. Sodium chloride has been demonstrated to contribute to some properties, especially the flavor of meat products and the inhibition of microorganisms (Kilcast et al., 2007). However, the high content of salt has been related to hypertension and cardiovascular disease (Doyle and Glass, 2010). A low content (4%) of sodium chloride was developed as an alternative concentration by many enterprises and factories in the meat industry. However, the effect of lower curing salt content on the non-volatile compounds of Chinese traditional dry-cured goose is not very clear. The present work focused on the mechanism and accumulation of total content of free amino acids with different dry-cured salt content. Therefore, we studied the degradation of myofibril proteins and the changes in potential proteolytic enzyme activities throughout the whole processing of Chinese traditional dry-cured goose. MATERIALS AND METHODS Processing of Dry-cured Goose and Sampling Twenty-four Eastern Zhejiang White Geese 75 d old with a live body weight of 3411 ± 73 g were purchased from a local processing plant. They were slaughtered in a commercial abattoir. The geese were randomly divided into 2 equal groups, then all of the whole carcasses were dry-cured by 2 different kinds of salt contents and placed in incubator at 4 C for 24 h. Two different salt levels were established: one group of 12 geese was dry-cured by adding 4% salt (w/w) (low salt level [LS]), whereas another group of 12 geese was dry-cured with 8% salt (w/w) (high salt level [HS]). After finishing the dry-cured procedure, geese were marinated in brine, which was comprised of 26% salt, 0.01% fennel, 0.02% ginger, and 0.03% shallot at 4 C for 24 h. Then, geese were air dry-ripened at 16 C at 68% relative humidity for 7 d. The flow speed of the air was maintained at about 6 m per s. The central parts of bicep femoris muscles were sampled at different processing points (raw meat, end of dry-curing, end of marinating, 3 d of dry-ripening, and 7 d of dry-ripening) for proteolysis enzymes, the degradation of myofibril proteins, and free amino acids analysis. Samples were wrapped in aluminum foil, frozen, and stored in liquid nitrogen until analysis. All of the determination was finished within 5wk. Preparation of Crude Enzyme Solution The crude enzyme solution was extracted as described previously by Hernández et al. (2004) with slight modification (n = 12). Biceps femoris muscle (0.5 g) was homogenized in 2.5 ml of 50 mm sodium citrate buffer containing 1 mm EDTA and 0.2% (v/v) Triton X-100 at ph 5.0. The extracts were homogenized (3 10 s at 10, 000 rpm while cooled on ice) with a DY89-I high-speed homogenizer (Scientz co., Ningbo, China) and centrifuged at 12, 000 g for 20 min at 4 C with a refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Changsha, China); the supernatant was filtered through glass wool and used for enzyme assays. The protein content of the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA). Cathepsin B+L Activities Determination The potential cathepsin B+L activities were assayed as described by Fidel Toldrá and Etherington (1988) using N-CBZ-L-phenylalanyl-L-arginine-AMC as substrates (n = 12). The reaction mixture consisted of 50 μl of enzyme solution and 250 μl of reaction buffer, 40 mm sodium phosphate at ph 6.0, containing 0.4 mm EDTA, 10 mm cysteine, and 0.05 mm of the specific substrate. Thereactionmixturewasincubatedat37 C; the fluorescence was continuously monitored at 355 nm and 460 nm as excitation and emission wavelengths, respectively, using with a 96-Well Plate Reader M200 (Tecan, Austria, Shanghai). Results were calculated as arbitrary intensity of fluorescence per microgram of protein at 37 C. Calpains Activities Determination The measurement of potential calpains activities was done as described previously by Soh et al. (2013) with slight modification (n = 12). This assay was performed with succinyl- Leu- Leu- Val- Tyr- 7- amino- 4- methylcoumarin (Suc- LLVY- AMC) substrate. The reaction mixture consisted of 50 μl of crude enzymes solution and 250 μl of reaction buffer, 100 mm Tris HCl, 1 mm DTT at ph 7.5, containing 0.05 mm of the specific substrate. The reaction mixture was incubated at 37 C, and the fluorescence was continuously monitored at 355 nm and 460 nm as excitation and emission wavelengths, respectively, using a 96-Well Plate Reader M200 (Tecan, Austria). Results were calculated as arbitrary intensity of fluorescence per microgram of protein at 37 C. Myofibril Proteins Preparation and Sodium Dodecyl Sulfate polyacrylamide Gel Electrophoresis (SDS PAGE) The extraction of myofibril proteins was performed according to the methodology described by Diaz et al.

3 2162 ZHOU ET AL. (1997) (n = 3). The proteolysis was determined by SDS- PAGE using a Mini Protean II apparatus (Bio-Rad, CA, Hercules) with 5 and 12.5% polyacrylamide gradient gels. The samples (5 mg/ml) were dissolved in a sample buffer (2% SDS and 5% β-mercaptoethanol) and heated at 96 C for 5 min. Protein samples (50 μg) were loaded in per lane. The fragments of myofibril proteins in gel were identified according to the molecular weight of standard protein. The intensities of bands were quantified by the specialized software and a microcomputer (Quantity One Quantitation Software, Bio-Rad Laboratories, Philadelphia, PA). Actin was defined as the internal standard. The difference of protein bands was calculated by the relative ratio of the intensities of protein bands, accounting for the intensity of actin. Free Amino Acids Determination The extraction and derivation of free amino acids were performed as described by Shim et al. (2013) with a little modification (n = 12). Eight hundred milligrams of sample were taken from liquid nitrogen and homogenized in 3 ml 3% 2- Hydroxy- 5- sulfobenzoic acid. After centrifugation at 15, 000 g at 4 C for 10 min, the supernatant was neutralized with 0.85 M K 2 CO 3, centrifuged again as described above, and filtered through a 0.22 μm syringefilter (Millipore, Billerica, MA). An L-8900 automated amino acid analyzer (Hitachi High- Technologies Corp., Tokyo, Japan) was used. The analytical column was a Hitachi HPLC Packed column (Ion-exchange resin, 4.6 mm i.d., 150 mm length, 3 μm particle size; Tokyo, Japan) with sulfone (SO 3 ) groups as the active exchange site; the analytical detector was a visible detector (Hitach High-Technologies Corp.). It was set to measure specific wavelengths of 570 and 440 nm for proline. Whole package and 4 prepared buffer solutions (Mitsubishi, Tokyo, Japan) were used as the mobile phase. The buffer solutions consisted of 4 different ph values (3.3, 3.2, 4.0, and 4.9). The regeneration solution and HPLC-grade water were used to clean and generate the analytical column. These 2 solutions were purchased from Mitsubishi (Tokyo, Japan). The above mentioned 5 types of buffers and water were used as a mobile phase using the gradient elution mode. The flow rate of the amino acid analyzer was 0.45 ml/min and the injection volume was 20 μl. The ninhydrin reagent, which contained sodium borohydrate and propylene glycol monomethyl ether, was purchased from Wako (Osaka, Japan). The buffer solution that contained lithium acetate dihydrate, glacial acetic acid, and propylene glycol monomethyl ether also was purchased from Wako (Osaka, Japan). These 2 solutions were used for the post-column reaction of analytes. During the running of the samples, the separate delivery pump for the ninhydrin reagent automatically mixed these 2 solutions, which were kept under nitrogen in the amino acid analyzer. The flow rate for the ninhydrin solution was 0.25 ml/min. Absolute quantification was defined using peak area with external standards and an internal control. Statistical Analysis Statistical analyses were carried out by the one-way analysis of variance (ANOVA) procedure (Duncan s Multiple Range Test) of SAS 8.0 software (SAS Institute Inc., Cary, NC) to analyze the changes of calpains, cathepsin activities, SDS-PAGE bands, and free amino acids during processing; HS and LS were classed in ANOVA analysis. The significant difference between HS and LS was analyzed by Student s t test model; processing points were classed in Student s t test. The statistically significant level was set as RESULTS AND DISCUSSION The Changes of Cathepsin B+L Activities The results of the changes of potential cathepsin B+L activities during the whole processing phase are shown in Figure 1. For the HS group, the potential activities of cathepsin B+L first decreased at the end of dry-curing then increased at the end of marination, and reduced at the third d of air dry-ripening and the end of air dryripening, compared to the raw (P < 0.001). For the LS group, the activities of cathepsin B+L decreased at the end of dry-curing and marinating then increased at the third d of air dry-ripening and reduced at the end of air dry-ripening, compared to the raw (P < 0.001). There was no significant difference of cathepsin B+L activities between the 2 groups throughout the whole processing stages, except for the end of dry-curing (P < 0.05) and the third d of air dry-ripening (P < 0.05). Residual cathepsin B+L activities in the Figure 1. Changes in cathepsin activity of dry-cured goose during processing (n = 12) A E Identical letters in the same line indicate that there is no significant difference in different process (P > 0.05). a-b Identical letters indicate that there is no significant difference in different groups (P > 0.05).

4 THE FORMATION OF TASTED COMPOUNDS OF DRY-CURED GOOSE 2163 final products for the LS and HS groups remained 73.0 and 69.6%, compared to the raw. The tendency of changes in potential cathepsin B+L activities during curing and dry-ripening in our results is very similar to the report of Zhao et al. (2005). Endogenous enzymes are the key enzymes for the degradation of cytoskeletal proteins in conditioning meat and dry-cured meat products (Wang et al., 2014). Cathepsins and calpains have been considered as the possibly crucial endopeptidases for the proteolysis of drycured meat products. However, overwhelming results demonstrated that cathepsin D lost its activity quickly, while cathepsin B, L, and H always kept some activities during dry-cured ham processing (Sárraga et al., 1993; Toldra et al., 1993). Ouali et al. (1987) suggested that cathepsin H hardly hydrolyzes any myofibril proteins, while cathepsin B and L possess extensive hydrolyzing activities for myofibril proteins. Cathepsin B and L are considered to be the main endopeptidases responsible for muscle proteolysis and flavor formation in dry-cured meat products, since they can keep relative stability during processing (Fidel Toldrá and Flores, 1998). In our results, the residual cathepsin B+L activities in the final products could cause the proteolysis of dry-cured goose meat. It is worth noting that the potential proteolytic activities were measured under optimum ph conditions. In the actual goose meat, the real cathepsin B+L activities were lower than those in vitro. The influence of salt content on potential cathepsin B+L activities has been reported during Chinese Jinhua ham and Spanish dry-cured ham processing (Garcia-Garrido et al., 2000; Zhao et al., 2005). Zhao et al. (2005) found that salt content exhibited a very weak impact on cathepsin B+L activities below 10 C. On the contrary, other reporters demonstrated that the activities of cathepsin B+L were strongly inhibited by chloride salts in drycured ham (Garcia-Garrido et al., 2000). Our results show that the LS group had higher cathepsin B+L activities at the end of dry-curing and the third d of air dry-ripening. This means that cathepsin B+L activities were inhibited by higher salt levels at the specific stages. However, cathepsin B+L activities in other processing points cannot be inhibited by high salt content. In addition, it had a fluctuation in cathepsin B+L activities during processing. The fluctuation could be attributed to salt might disrupting the lysosome membrane which prevented the inhibition of cathepsin B+L activities. Changes in Calpains Activities The changes in calpains activities during the whole dry-cured goose processing phase are shown in Figure 2. For both groups, the activities of calpains reduced at the dry-cured, marinated stage, and the third d of air dry-ripening (P < 0.01 for the HS group; P < 0.05 for the LS group), and then remained about half of activi- Figure 2. Changes in calpain activities of dry-cured goose during processing (n = 12) A D Identical letters in the same line indicate that there is no significant difference in different process (P > 0.05). a b Identical letters indicate that there is no significant difference in different groups (P > 0.05). ties of those in raw meat, at the end of air dry-ripening phase (53.6% in HS group; 55.6% in LS group). There were higher calpains activities in the LS group at the end of the dry-cured stage (P < 0.05) compared to the HS group; there was no significant difference between the 2 groups during other stages. The calpains have been shown to be an important contributor to proteolytic degradation of myofibril proteins during tenderizing (Koohmaraie and Geesink, 2006). Pomponio and Ertbjerg (2012) reported that μ-calpain and m-calpain degraded myofibril proteins and improved the tenderness of raw meat. Melody et al. (2004) implied that the appearance of the autolysis fragments of calpains is indicative of their activation. Pomponio et al. (2008) demonstrated that not only μ-calpain, but later also m-calpain has potential to autolyze during postmortem storage in porcine muscle, suggesting that the sarcoplasmic calcium level under certain time-temperature combinations rises to the level required for activation of μ-calpain and later also m-calpain. Geesink et al. (2006) reported that the changes of m-calpain activity had significantly enhanced after 3 days of refrigerated storage in murine hind limb muscles. The influence of salt content on calpain activities has been reported during dry-cured processing. Rosell and Toldrá(1996) demonstrated that the activities of calpains were inhabited by a high sodium chloride level. The decrease during processing and the difference of calpains activities at the end of dry-curing between HS and LS groups in our results could be explained by the addition of dry-cured salt. Sárraga et al. (1993) reported that calpains activities decreased to to 72.61% after salting, and the retained activities were dependent on muscles and the kinds of animals; they suggested that the early disappearance of calpains activities in the curing could be explained by the instability of enzymes, especially calpain I. Rosell and Toldrá (1996) indicated that no

5 2164 ZHOU ET AL. calpains activities were detected after 20 d of dry-curing during the processing of dry-cured ham. Sárraga, et al. (1989) suggested that a long-time curing process and the addition of curing salt made calpains activities unlikely to be involved during the whole processing of Spanish dry-cured ham. In our results, the highly retained calpains activities compared to the raw were assumed to be relative to the special kind of birds and the shorter curing time than dry-cured hams. Degradation of Myofibril Proteins Figure 3 shows the SDS-PAGE results of myofibril proteins with different dry-cured salt content during processing. The cleaved bands of myosin light chain (MLC) and troponin-i were identified during the processing in both HS and LS groups. The relative intensities of troponin-i and MLC bands normalized with actin in HS and LS are shown in Figure 4. There was significant different relative intensity of troponin-i at the third d of air dry-ripening, but no difference of Figure 3. SDS-PAGE electrophoresis of myofibril proteins during dry-cured goose processing. Figure 4. Relative intensities of troponin-i and MLC bands normalized with actin in HS and LS groups during dry-cured goose processing (n = 3). A C Identical letters in the same salt content group indicate that there is no significant difference in different processing points (P > 0.05). a b Identical letters indicate that there is no significant difference in different salt content groups (P > 0.05). troponin-i and MLC was observed in the final products. Several protein fragments have been identified in drycured meat products by proteomic tools, such as actin (Sentandreu et al., 2007), titin, MLC (Mora et al., 2009), creatine kinase (Mora et al., 2010), pyruvate kinase (Sforza et al., 2003), troponin proteins (Mora et al., 2010), et al. Information derived from the identification of the protein fragments naturally generated could be very important to better understand the mechanism s flavor development during processing. Toldra et al. (1993) found a progressive disappearance of MLC 1 and MLC 2 during processing in biceps femoris muscle of dry-cured ham. Wang et al. (2014) reportedthe C-proteins, β-tropomyosin, troponin-i, and troponin- C were cleaved in the processing of dry-cured duck; they reported the significant correlations among the changes of the degraded bands of proteins, free amino acids content, and cathepsin activities. In our results, the main hydrolyzed proteins were MLC and troponin- I. The combined influence of retained activities of calpains and cathepsin B & L played the key role in the degradation of myofibril proteins. Changes in Free Amino Acids The evolution of 18 free amino acids (FAA)andthe total free amino acid (TFAA) contents along with the processing of dry-cured goose is shown in Table 1. Compared to raw meat, the TFAA contents significantly increased by and 31.82% in the final products for HS (P < 0.001) and LS (P < 0.01), respectively. The main FFA were tryptophane, alanine, glutamic acid, leucine, taurine, and lysine. The TFAA did not have a significant difference throughout the whole processing steps and in the final products, except at the third d of dry-ripening (P < 0.001) between the HS group and LS group. It has been reported that peptides and free amino acids from protein degradation products are the precursors of flavor compounds (Toldra, 1998). The changes in FAA have been studied in dry-cured ham (Prevolnik et al., 2011; Bermúdez et al., 2014). The non-volatile compounds generated from proteins may be of importance in the sensory quality of dry-cured goose. The increase in TFAA could be attributed to the proteolysis of MLC and troponin-i, since Wang, et al. (2014) demonstrated the significant correlations among the changes of the degraded proteins, free amino acids contents, and proteolysis activities. The results that no significant difference on TFAA between the HS group and the LS group was observed in the final products do not agree with those obtained by Virgili et al. (1999), who found that the free amino acid content was negatively correlated with salt content. On the contrary, Martín, et al. (2001) reported that hams from traditional processing with high salt

6 THE FORMATION OF TASTED COMPOUNDS OF DRY-CURED GOOSE 2165 Table 1. Changes in free amino acids content (mg/100 g) of dry-cured goose during processing (n = 12). Freeamino Rawmeat Endofdry-curing Endofmarinating Thirddendof Seventhdendof acid (0d) (1d) (2d) drying-ripening (5d) drying-ripening (9d) Group HS LS HS LS HS LS HS LS HS LS Asp 2.55 A,a 2.08 A,a 2.74 A,b 2.23 A,a 2.06 A,a 1.26 A,a 3.24 A,a 6.49 B,b 7.90 B,a 5.38 B,a Thr 6.98 A,a 6.87 B,a 5.49 A,a 6.99 A,b 5.25 A,a 4.64 A,a 6.48 A,a 8.78 B,a 8.99 B,a 8.85 B,a Ser 9.12 A,a 9.77 A,a 8.03 A,a 9.89 A,a 6.79 A,a 7.21 A,a 8.44 A,a B,a B,a B,a Glu 9.37 A,a A,a A,b 9.08 A,a A,a 8.41 A,a A,b A,a B,a B,a Pro 4.85 A,a 2.93 A,a 3.52 A,a 4.63 A,b 3.43 A,a 3.02 A,a 4.72 A,a 5.75 B,a B,a 6.44 B,a Gly 6.39 B,a 6.64 B,a 6.46 B,b 3.94 A,a 3.39 A,a 4.16 A,a 4.98 A,a 6.74 B,a B,a 6.79 B,a Ala B,a B,a B,b A,a A,a A,a A,a B,a B,a B,a Val 7.74 A,a 6.26 B,a 7.14 A,b 5.80 A,a 5.53 A,a 4.61 A,a 6.44 A,a 9.27 C,a 9.81 B,a 9.93 C,a Met 2.84 A,a 3.83 B,a 4.03 A,b 2.07 A,a 3.65 A,a 3.76 B,a 4.31 A,a 6.47 C,a 8.86 B,a 5.98 C,a Ile 5.07 A,a 4.22 A,a 4.50 A,a 5.27 A,a 4.46 A,a 4.17 A,a 5.33 A,a 9.63 B,b 7.28 B,a 8.23 B,a Leu 8.01 A,a 9.66 A,b 8.11 A,a 8.32 A,a 9.98 A,a A,a A,a B,b B,a B,a Tyr 4.57 A,a 5.94 A,a 4.24 A,a 4.46 A,a 5.94 A,a 5.37 A,a 6.50 A,a B,b B,a B,a Phe 4.77 A,a 5.25 A,a 4.22 A,a 5.08 A,a 6.75 A,a 6.39 A,a 7.51 A,a B,b B,a B,a Trp A,a A,a A,b B,a B,a B,a A,a B,b B,a B,b His 3.23 A,a 2.92 B,a 3.76 A,b 2.50 A,a 2.85 A,a 2.53 A,a 3.75 A,a 5.14 C,a 5.37 B,a 5.51 C,a Lys 5.85 A,a 6.80 B,a 6.69 A,b 5.51 A,a 7.54 A,a 6.08 B,a A,a C,a B,a C,a Arg 5.44 A,a 5.75 A,a 6.15 A,a 6.10 A,a 7.23 A,a 6.49 A,a 9.31 B,a B,b B,a B,a Tau B,a B,a A,B,a B,a A,a A,a A,a A,a A,a A,a total A,a A,a A,a A,a A,a A,a A,a B,b B,a B,a A C Identical letters in the same salt content indicate that there is no significant difference in different processing points (P > 0.05). a,b Identical letters indicate that there is no significant difference between HS and LS groups (P > 0.05). content had higher contents of free amino acids than hams from modern processing with low salt content. The different results could be related to the different styles of products. In Chinese traditional dry-cured goose, there was no difference in proteolysis activities and MLC and troponin-i bands in final products between groups. This means that 4% low-salt dry-curing had no negative effect on the eating characteristics of the final product. CONCLUSION The increase of TFAA could be attributed to the proteolysis of myofibril proteins and the combined influence of retained activities of calpains and cathepsin B+L. The different proteolysis activities during processing did not cause the different MLC and troponin-i bands and TFAA contents in final products between groups. It seems that 4% low-salt dry-curing can be used in future applications. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China ( ), Modern Agricultural Technical System Foundation (CARS-43-17), National Agricultural Transformation of Scientific and Technological Achievements Projects of China (2013GB2C200191), the Scientific and Technological program of Ningbo University (XKL14D2087 and 2013C910017), and the K.C. Wong Magna Fund at Ningbo University. REFERENCES Bermúdez, R., D. Franco, J. Carballo, M. Á. Sentandreu, and J. M. Lorenzo Influence of muscle type on the evolution of free amino acids and sarcoplasmic and myofibrillar proteins through the manufacturing process of Celta dry-cured ham. Food Res. Int. 56: Diaz, O., M. Fernandez, G. D. G. De Fernando, L. de la Hoz, and J. A. Ordoñez Proteolysis in dry fermented sausages: the effect of selected exogenous proteases. Meat Sci. 46: Doyle, M. E., and K. A. Glass Sodium reduction and its effect on food safety, food quality, and human health. 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